Method of diagnosing changes in the intestinal absorptive surface in an individual

ABSTRACT

The invention relates to a method of diagnosing changes in the intestinal absorptive surface in an individual. According to the invention, an immunoassay is performed on samples of serum and/or plasma from the blood of the patient in order to determine the quantity of human apolipoprotein apoA-IV using a specific antibody obtained from purified human intestinal apoA-IV (purified from human plasma), recombinant human apoA-IV or an active fragment of human apoA-IV, said assay being of the immunoelectrophoresis-, ELISA- or Western blot-type immunoassay. The invention is advantageous over standard clinical methods (intestinal biopsy and endoscopy) owing to the fact that it is non-invasive for the patient and that it can be easily industrialised. Said method can be used to diagnose functional intestinal disorders caused by an abnormal amount of functionally-effective intestinal absorptive surface, such as malabsorption, coeliac disease, villous atrophy, short bowel syndrome, chronic diarrhoea and flatulence.

This invention refers to new diagnostic methods and kits in the field ofclinical gastroenterology, which are based on the assessment of anatural intestinal protein in a sample of plasma and/or serum removedfrom a patient.

BACKGROUND ART

Several intestine function disorders which are totally or partiallycaused by absence (e.g. because a loss or a lack of maturity) offunctionally effective absorptive intestinal mass are malabsorption,coeliac disease, villus atrophy, short bowel syndrome, chronic diarrheaand flatulence, being malabsorption one of the most prevalent ones.Malabsorption (i.e. impaired intestinal absorption of nutrients, mainlysugars and lipids) is a patology associated with several physiologicalconditions, such as malnutrition, aerophagia, gastrointestinaldisturbances and general disconfort. One of the main causes ofmalabsorption is intestinal atrophy or decrease of intestinal mucosasurface (i.e. decrease of the whole absorptive mass of intestinalvilli).

In relation with therapy of different intestine function disorders, itis interesting to diagnose the status of whole functionally effectiveabsorptive mass of a given patient, either at various stages of hisindividual development (i.e. at his various ages), or along atherapeutical treatment of intestinal failure condition. However, mostdiagnostic methods used today for this purpose are aggressive and basedeither on endoscopy or on analysis of jejunal biopsy specimens.

Most clinicians diagnose malabsorption syndromes by endoscopy andintestinal biopsy, but these practices have some drawbacks andlimitations. Thus, on several occasions intestinal biopsy is notrepresentative of the status of the whole intestine and a mucosa mosaicis observed with areas of normal and atrophied villi (cf. S. C. Tawil etal., “Scalloping of the valvulae conniventes and mosaic mucosa intropical sprue”; Gastrointest. Endosc. 1991, vol. 37, pp. 365-367).Besides, the orientation of biopsy may occasionally hinder diagnosis, asa result of which partial intestinal atrophy may not be detected (cf. M.Cassaro et al., “The dark side of the gastric biopsy”, Hum. Pathol.1999, vol. 30, pp. 741-744). About half of those who suffer from celiacdiseases (nutritional intolerance to gluten) are not diagnosed sincethey do not show characteristic symptoms of malabsorption; and the sameoccurs with tropical sprue (tropical stomatitis). Chromoendoscopicstudies, which show magnifications of the intestinal mucosa duringendoscopy, reveal that a significant number of partial atrophies wouldnot have been detected by classic endoscopy (cf. L. M. Siegel et al.,“Evaluation of latino expatriates for evident of tropical sprue(abstract)”; Gastroenterology 1995, vol. 108, p. A324). Therefore, itwould be useful to find serum and/or plasma markers that indicate theclinic status of the intestinal mucosa, particularly intestinal atrophy,and thus obviate the general use of invasive methods like jejunal biopsyand endoscopy.

In clinical field, in order to diagnose intestinal malabsorption, a fewmethods in serum and/or plasma different from classic biopsy andendoscopy have been suggested in the art. According to one of thesemethods, lipid malabsorption in patients with chronic pancreatitis isdiagnosed by an indirect test that measures the apolipoprotein B-48(apoB-48) in plasma after a meal rich in lipids (cf. B. Saviana et al.,“Diagnosis of lipid malabsorption in patients with chronic pancreatitis:a new indirect test using postprandial plasma apolipoprotein B-48”, Am.J. Gastroenterol. 1999, vol. 94, pp. 3229-3235). This method implies theprevious isolation of lipoproteins, what is a source of limitations forclinical practice (high volume of blood sample, special and expensiveequipment, loss of said lipoproteins during quilomicra isolation, etc.).Another method is based on the measurement of circulating citrulline byHPLC (high performance liquid chromatography) as a marker ofmalabsorption in cases of short-bowel syndrome (cf. P. Crenn et al.,“Postabsortive plasma citrulline concentration is a marker of absorptiveenterocyte mass and intestinal failure in humans”; Gastroenterology2000, vol. 119, pp. 1496-1505). A major limitation of this method isthat it can be used only with adults, because the onset for citrullinesynthesis in the enterocyte takes place after weaning of mammals (cf. G.Wu et al., “Synthesis of citrulline from glutamine in pig enterocytes”;Biochemical J. 1994, vol. 299, pp. 115-121). Measurement of sugar inurine and serum by HPLC following an oral load of these sugars(lactulose, mannitol, lactose, D-xylose . . . ) has also been proposedfor studying malabsorption in adults and children (cf. S. Travis et al.;“Intestinal permeability: functional assessment and significance”; Clin.Sci. 1992, vol. 82, pp. 471488. J. A. Kynaston et al., “Simultaneousquantification of mannitol, 3-O-methyl glucose, and lactulose in urineby HPLC with pulsed electrochemical detection, for use in studies ofintestinal permeability”; Clin. Chem. 1993, vol. 39, pp. 453456). But itis frequently annoying for the patient, and it is besides depending ofother factors such as gastric emptying and the rate of urinerecollection. With paediatric patients, other more indirect methodsused, which are also annoying and besides give false positives, are e.g.the breath tests which involve measurement of expired hydrogen producedby bacteria that ferment non absorbed hydrocarbons, or measurement ofexpired radiactive ¹⁴CO₂ produced by the metabolism of the ¹⁴C-trioleineingested, which is related to malabsorption of lipids (cf. F.Fernandez-Bañares et al., “Procedimientos diagnósticos engastroenterologia y hepatologia”, in P. Humbert & M. A. Gassull (eds.),Capitulo 8, pp. 71-79; Mosby-Dioma Libros S. A.; Madrid 1995).

Thus, it is of clinic interest to find new diagnostic methods that, in agiven patient, indicate the status of the whole functionally activeabsorptive intestinal mass, particularly in relation to severalintestine function disorders.

SUMMARY OF THE INVENTION

According to an aspect of the present invention, it is provided a methodof diagnosis of an intestine function disorder caused by an abnormallevel of functionally effective absorptive total mass in a givenpatient, comprising submitting serum and/or plasma samples frompatient's blood to an immunoassay for assessing the amount of humanapolipoprotein apoA-IV. The immunoassay involves an specific antibodyanti-human apoA-IV obtained from a purified intestinal human apoA-IV, arecombinant human apoA-IV or an active fragment of human apoA-IV.

Apolipoprotein A-IV (apoA-IV) is an approximatedly 46 KDa proteinsynthesised by enterocytes (the most abundant cells of intestinalepithelium), it is released to the lymph bound to chylomicra(lipoproteins synthesised after a meal rich in lipids), and thence it isreleased to the bloodstream where it is present mainly in free form.ApoA-IV is expressed in human from week 20 of gestation, what means thatit can be quantified at birth and prenatal ages. There are presentlyeight known isoforms (genetically determined variants) of human apoA-IV.Depending on the population examined, the allele frequency of theprimary isoform is about 94%; the second most prevalent isoform is about6%. The other identified isoforms having a very low frequency. ByapoA-IV it is here meant any of its isoforms or their mixtures.

The method of the present invention makes use of known assays fordetection and quantification of human apoA-IV in serum and/or plasma.Immunodiffusion assays by electrophoresis, such as those disclosed in EP282.412 B1 for other apolipoproteins, can also be used in the method ofthe present invention. However, ELISA (Enzyme-linked immunosorbentassays) and Western blot assays are preferred.

The absorptive intestinal mass which is functionally effective in thepatient refers to the part of its intestinal mucosa which is mainlyformed by enterocytes (absorptive cells). An abnormal level offunctionally effective absorptive intestinal mass may be the result of aloss of enterocytes (e.g. by ethanol intake), or may be caused by a lackof maturity of the cells (e.g. in premature neonates). By specificantibody anti-human apoA-IV it is meant any monoclonal or polyclonalantibody which interacts only with human intestinal apolipoproteinapoA-IV. This antibody can be obtained by any of the standard methodsknown by persons skilled in the art, using as immunogen either purifiedhuman apoA-IV (in any of its isoforms or mixtures thereof), or arecombinant human apoA-IV, or an active fragment of human apoA-IV.

In a preferred embodiment, the immunoassay used in the method of thepresent invention is of the ELISA sandwich type. In a more preferredembodiment, the ELISA sandwich immunoassay involves the pretreatment ofthe sample with a specific buffer (clearing buffer) containing apancreatic cholesterol esterase for decreasing interference of samplecirculating lipids in apoA-IV quantification. In another preferredembodiment the immunoassay is a Western blot assay (specially preferredwith an internal control). And in another preferred embodiment, theimmunoassay is an immunodiffusion assay.

Among the intestine function disorders caused by an abnormal level offunctionally effective absorptive intestinal mass, which can bediagnosed by the method of the present invention, there aremalapsorption, celiac disease (i.e. intolerance to gluten of the diet),intestine villus atrophy, short bowel syndrome, chronic diarrhea andflatulence. Among the different causes of these intestinal disordersare: genetic predisposition, malnutrition, fasted state, parenteralnutrition, intestinal surgery, chemotherapy, premature birth, intestinalbacterial infection, etc.

According to another aspect of the present invention, it is provided anin vitro kit for the diagnosis of an intestine function disorder causedby an abnormal level of functionally effective absorptive intestinalmass (including any of the above-mentioned conditions) in a givenpatient. The kit has an specific antibody anti-human apoA-IV obtainedfrom a purified intestinal human apoA-IV, from a recombinant humanapoA-IV or from an active fragment of human apoA-IV, together with othermaterials and/or components for assessing the amount of humanapolipoprotein apoA-IV by immunoassay. In preferred embodiments, the kithas materials and/or components for an ELISA type immunoassay, or forWestern blot immunoassay, or for immunodiffusion assay. For a personskilled in the art it will be easy to select materials and/or componentsfor preparing in vitro kits appropriate for the specific immunoassay,e.g. a microplate with wells in an ELISA, or a film covered with gel ina Western blot or in an immunodifussion assay.

The assessment of the amount of human apolipoprotein apoA-IV can be moreor less quantitative depending on the kind of immunoassay used and theavailability of pure human apoA-IV as standard, as illustrated in theaccompanying examples.

It is noteworthy that several factors such as dietary fat intake, fatmalabsorption associated to pancreatic disorders, acute inflammation,and hormonal dysregulation can disturb the plasma apoA-IV concentrationin humans. Thus, there are few pathologies associated with lipidmetabolism in which these plasma concentrations are altered. In patientswith these pathologies the skilled person should use the method of thepresent invention carefully, taking the blood sample at least afterovernight fast. Although absolute levels of apoA-IV in patients withthese pathologies can differ significantly from average values of thecontrol population, the evolution of the apoA-IV level for a givenpatient will still be meaningful. Among these pathologies are thefollowing: coronary disease, obesity, diabetes mellitus, renalinsufficiency, chronic pancreatitis and severe undernourishment.

So far human apoA-IV has received little attention in the fields ofclinic diagnosis and therapy, an exception being the suggestion ofadministering human apoA-IV to patients as an injectable formulation forthe purpose of controlling appetite and reducing food intake and bodyweight (cf. WO 94/27629 A1).

Compared with the clinical methods current in the art (endoscopy andjeyunal biopsy), the invention is advantageous in that it provides adiagnostic method which is not invasive to the patient, and that can beeasily industrialized with samples of plasma and/or serum removed fromthe patient.

Compared with the possible assessment of other intestinal proteins,another advantage of this invention arises from the fact that humanapoA-IV can be assessed at any patient age of the individual, includingprenatal stages. The fact that half life of human apoA-IV is low and itsconcentration in plasma significantly correlates with its release ratefrom enterocyte, not with its degradation rate, makes the assessment ofhuman apoA-IV very indicative of the mucosa functional and morphologicalstatus. A further advantage, compared with the assessment of otherapolipoproteins, arises from the fact that human apoA-IV is released toblood stream mostly in a free form (i.e. non-associated tolipoproteins), what results in that the method of the present inventionsuffers less from interferences with circulating lipids.

Throughout the description and claims the word “comprise” and itsvariations are not intended to exclude other additives, components,integers or steps. The disclosure in the abstract is incorporated hereinas reference. The following detailed description and drawings areprovided by way of illustration, and they are not intended to belimiting of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the detection of human apoA-IV in plasma andintestinal biopsy by Western blot assay.

FIG. 2 shows a histological immunolocalisation of apoA-IV in a humanintestinal biopsy of duodenum-proximal jejunum.

FIG. 3 includes two graphs of an ELISA sandwich for detecting apoA-IV inhuman plasma.

FIG. 4 corresponds to two Western blot assays for purified apoA-IV andhuman plasma, throughout pre and postnatal development.

FIG. 5 shows apoA-IV levels by ELISA sandwich assay in umbilical cordserum of human neonates, from alcoholic and control mothers, accordingto the distribution's body weight.

FIG. 6 corresponds to a Western blot assay to detect apoA-IV inumbilical cord serum of human neonates, from alcoholic and controlmothers.

FIG. 7 shows detection and quantification by Western blot assay ofplasma apoA-IV in coeliac children at different moments of theirdisease.

FIG. 8 shows detection and quantification by Western blot assay ofplasma apoA-IV, in parenterally nourished patients after abdominalsurgery.

FIG. 9 shows detection and quantification by Western blot assay ofplasma apoA-IV in successive parenteral-(fast state) and enterallynourished patients after gastric surgery.

DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS

The method of the present invention, by using both ELISA sandwich andWestern blot assays, is illustrated bellow in two representativepaediatric pathologies with intestinal failures (foetal alcohol effectsand coeliac disease) and in two intestine atrophy models in adults(parenteral nutrition and fast-refeed nutrition). In rat model,inventors have previously shown that etanol consumption by the motherduring gestation causes intestinal disorders in foetus and new-born (cf.L. Camps et al., “Effects of prenatal exposure to ethanol on intestinaldevelopment of rat fetuses”; Journal Pediatric Gastroenterol. Nutr.1997, vol. 24, pp. 302-311, and references therein), and some clinicalcases in humans have also been published (cf. S. Tourtet et al., “Smallintestine atresia and abnormal insertion of the umbilicus in a childwith fetal alcohol syndrome”; Archives de Pediatrie 1997, vol. 4, pp.650-652, and references therein). Coeliac disease (intolerance to glutenof the diet) is a well-established pathology of severe or moderateintestinal villous atrophy, for which the definitive diagnosis is doneso far through intestinal biopsy. It is well known that any parenteralnutrition and general fasted state develop mucosa disorder with someatrophy of intestinal villi.

Population Groups of the Four Models

Babies born from mothers who had consumed alcohol during pregnancy weredistinguished of babies born from control mothers: A woman was definedas an alcoholic mother if she had taken alcohol during gestation andbefore delivery, trough an interview and specific tests.

The children diagnosed as coeliac had endomysium antibodies in the serumand took a diet including gluten. The coeliac children who had a jejunalbiopsy (almost the 80%) had intestinal atrophy in all cases. The age ofstudy was between 1-6 years-old.

Parenteral nutrition was performed on adult and older patients (30-80years-old) submitted to abdominal surgery (colon resection). Allpatients were selected according the following criteria: no kidney orliver disease, no peritoneal carcinomastosis or known metastases, nomalnutrition, no weight loss, and no metabolic or endocrine disease.After the operation patients receiving parenteral nutrition support,without oral intake until day seven.

Other groups of patients (submitted to gastric resection) and selectedwith the same criterion were on parenteral nutrition (tree or five daysafter surgery) and on enteral nutrition (with liquid and soft dietduring four days after) successively.

Clinical Samples

Plasmas and/or serums from patients were taken after overnight fast,except for neonates. At birth serum samples from umbilical cord bloodwere collected in corresponding Obstetrics and Gynaecology Departments.Plasmas were obtained in disodium EDTA and then centrifuged and frozenat −30° C.

A small fragment of intestinal biopsy was placed at 4° C. in PBS(Phosphate Buffer Saline, pH 7.5) and processed as quickly as possiblefor Western blot and/or histological immunolocalisation studies.Biopsies for western analysis were processed previously at 4° C. and toa dilution of 1/25 with PBS-Tritonx100 at 1% (v/v).

Clinical studies were performed with agreement of Ethical Committees ofdifferent Hospitals involved: Hospital Clinic de Barcelona, HospitalSant Joan de Déu in Barcelona, and Hospital Vall d'Hebron in Barcelona.

Western Blot to Detect ApoA-IV in Plasma, Serum and Intestinal Biopsy

The Western blot assays to detect apoA-IV used the proteins separated in10% SDS-PAGE (sodium dodecylsulfate-polyacrylamide gradientelectrophoresis) and transferred on membranes of “Immovilon-P”,Millipore®. Aliquots of diluted serum and/or plasma (1/100) were loadedwith sample buffer, resulting 0.2 μl of serum and/or plasma per well.The anti-human apoA-IV IgG (developed in inventors' laboratory) obtainedfrom rabbit at 1/500 dilution was used as primary antibody; and anti-IgGfrom commercial rabbit obtained in pig (marked with horse radishperoxidase, HRP) at 1/15,000 dilution, as secondary antibody (Dako®,Glostrup, Denmark). Other special dilutions for antibodies are describedin the corresponding figures. Non-specific binding was blocked with BSA(bovine serum albumine) at 2% (w/v). To develop the Western blot the ECL(enhanced chemiluminiscence luminol) commercial system was used(Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, England)or SuperSignal West Pico chemiluminescent substrate for detection HRP(Pierce®, Rockford, Ill., USA).

ELISA Sandwich for Quantification of ApoA-IV in Serum and/or Plasma

ApoA-IV plasma concentration was determined by an ELISA (enzyme-linkedimmuno-absorbent assay) sandwich developed in inventors laboratory. 500ng of the antibody capture developed in inventors' laboratory (IgG ofanti-apoA-IV), obtained in rabbit and diluted in 100 μl ofcarbonate-bicarbonate buffer (6.9 g/l Na₂CO₃, 10.5 g/l NaHCO₃, pH 9.6),was immunoassayed on a polystyrene plate with 96 wells. After all-nightincubation of the plate at 4° C. in a humid chamber, it was washed fivetimes with PBS-T (Phosphate Buffer Saline pH 7.4, with 0.05% Tween-20,v/v) at room temperature. To block non-specific binding, each well wasincubated with 360 μl of blocking solution containing bovine serumalbumin (BSA) (PBS and 0.5% BSA, w/v) for one hour at 37° C. in a humidchamber and shaken. After being washed five times with PBS-T, 100 μl ofthe samples, the standard and the internal control (for inter- andintra-assays, see below paragraph) already diluted in PBS-T-BSA (PBS,0.5% BSA and 0.05% Tween-20), were incubated for one hour at 37° C. in ahumid chamber. Then it was washed five more times with PSB-T.

The plate was then incubated for 2 hours at room temperature in thehumid chamber with the antibody detector, i.e. the same human IgGapoA-IV antibodies obtained in rabbit, but bound to biotine (0.5 mg/ml,12.4 mol biotine/mol IgG), and diluted 1/500 in 100 μl PBS-T-BSA. Whenthe incubation was complete, the plate was washed five times with PSB-T.Then it was incubated for 20 min at room temperature in the humidchamber with a solution of avidine bound to 0.2 mg/l peroxidase anddiluted in PBS-T-BSA (100 μl per well). It was washed five times withPSB-T solution. Then 100 μl per well of OPD solution (0.4 g/lO-phenylendiamine, 7.3 g/l citric acid, 9.6 g/l Na₂HPO₄ and 0.2 μl/mlH₂O₂), prepared at moment of use, was pipetted. The development orappearance of the product's colour took place at room temperature. After20 min at most, the reaction was stopped by adding 50 μl per well of 2.5M HCl. The plate was shaken for a few seconds in an orbital shaker andwas read at 492 nm with the “Titertek Multiskan” ELISA reader.

Pre-treatment of plasmas or serums: In pilot experiments to fine-tuneELISA not totally satisfactory results were obtained: There was nolinearity in the volume of the samples; and inter- and intra-assayrepetition was unacceptable. The samples had to be pre-treated withpancreatic cholesterol esterase, avoiding possible interference ofplasma lipids in quantification of apoA-IV, in order to attain betterresults. The plasma samples and the internal control (for inter- andintra-assays) were diluted 1/10 in PBS and then incubated at 37° C. withclearing buffer used for nephelometry which contained: 87.2 g/l asparticacid, 17.5 g/l sodium cholate, 1.2% Tween 20, 0.4 g/l cholesterolesterase, and 0.1 M Tris, pH 7.7 in (1:1) (v:v). The reaction was haltedwith ice after ten minutes and then PBS-T-BSA was added, until the finaldilution of the sample (1/150 or 1/300) was attained. Clearing bufferwas processed without serum and/or plasmas as background. The standardwas made from an apoA-IV solution previously purified in inventorslaboratory. Protein concentration was confirmed with the commercial kit“BCA Protein assay reagent” (Pierce®, Rockford, Ill.), using BSA asstandard. The standard of apoA-IV was diluted in PST-T-BSA, using aconcentration range of 1 to 2,000 ng per well. It was established thatthere was no interference of the clearing buffer in the standard, andtherefore it was not used in it.

The standard curve was adjusted through polynomial regression analysis.Two dilutions of each sample were assayed (1/150 and 1/300) and theapoA-IV concentration was calculated from the mean of the two dilutions.The dilutions of the sample and the various concentrations of thestandard control were assayed in duplicate. The internal control was anadult plasma overnight fasted with a concentration of apoA-IV: 13.14mg/100 ml, and it was assayed in quadruplicate. The intra- andinter-assay was respectively 4.2% and 9.1%.

Detection of Human ApoA-IV in Plasma and Intestinal Biopsy by WesternBlot Assay.

The specificity of antibodies (from rabbit IgG) obtained by inventors,for detecting apolipoprotein apoA-IV in human plasma and intestinalbiopsy, is illustrated by results in FIGS. 1, 2 and 3.

FIG. 1 illustrates results obtained by SDS-PAGE electrophoresis andWestern blot. In FIG. A 30 μg of protein from plasmas (rat and humanorigin) were loaded in wells. A well was loaded with 20 ng of purifiedhuman apoA-IV. The primary antibody was diluted 1/200, and the secondaryantibody (anti rabbit IgG bound to HRP, horse radish peroxidase) to1/2,000. Detection was made by DAB (diaminobenzidine). It wasdemonstrated that the primary antibody do not label the apoA-IV fromrat. In FIG. B 22 ng of purified human apoA-IV and 5 μg of protein ofhuman plasma were loaded. Different protein contents of intestinalbiopsy (18 μg and 9 μg) were loaded in different wells. The primary andsecondary antibodies were processed identically to results of FIG. A.Detection was made by ECL. Detection by ECL showed higher label thandetection by DAB. FIG. 1A symbols are as follows: Pr=plasma from rat.Ph=plasma from human. ApoA-IV is the protein purified in inventors'laboratory. FIG. 1B symbols are as follows. Ih means human intestinalbiopsy (from duodenum-proximal jejunum).

Immunolocalisation of Apolipoprotein A-IV in a Human Intestinal Biopsyof Duodenum-Proximal Jejunum.

FIG. 2 shows immunolocalisation of apoA-IV in human intestine usingconfocal microscopy (Leica TCS 4D, Serveis Cientifico-Tècnics, ParcCientific, UB). The primary antibody (rabbit IgG) was diluted to 1/40 inPBS-FCS (fetal calf serum at 10%, v/v); as secondary antibody it wasused anti rabbit IgG obtained from goat bound to Texas red which wasdiluted 1/200 in PBS-FCS. The images showed that the protein was locatedalong the epithelium of the intestinal mucosa (see to 50 and 25 μm), andspecifically in enterocytes with no blot found in other kind of cells.ApoA-IV was mainly located in the supranuclear area of the enterocyte(see to 10 and 5 μm). It was shown the cellular location and specificityof inventors antibody (rabbit IgG) for detecting human apoA-IVsynthesised basically by enterocytes. These data supplemented previousresults obtained with Western blot assay in FIG. 1B.

ELISA Sandwich for Detecting Apolipoprotein A-IV in Human Plasma.

FIG. 3 shows different results of ELISA sandwich method. FIG. 3Aillustrates the specificity of inventors antibody for detecting growingconcentrations of purified human apoA-IV. Commercialised human apoA-I(synthesised also by the intestinal mucosa) and human albumin are notdetected using the same ELISA sandwich method. In FIG. 3B increasingvolumes of sample (dilution assayed) are correlated to human plasmaswith varying amounts of apoA-IV: low levels for neonates, high levelsfor a child of 5-6 years-old, and adult levels for an adult of 20years-old. The recuperation value of purified apoA-IV into differentsamples was between 90.5 and 106.2%. FIG. 3A symbols are as follows:human apoA-IV (squares), human apoA-I (triangles) and human albumin(circles). FIG. 3B symbols are as follows: plasma from a child of 5-6years-old (triangles), plasma from an adult of 20 years-old (circles),plasma from a neonate birth at term (rhombus).

Western Blot Assay for Purified ApoA-IV and for Detecting ApoA-IV inHuman Plasma Throughout Pre and Postnatal Development.

FIG. 4 shows different blots of human plasma with different amounts ofapoA-IV according the stage of development (from birth to adult ages).Western blot assay can be used as an alternative method to ELISAsandwich quantification. Detection limit of Western blot in the presentexperimental conditions is about 5-2 ng of protein apoA-IV and 0.2 μl ofserum and/or plasma sample. Correlation index of values from 59 samplesparallel measured by both analysis was 0.841. Although the Western blotassay is a semi-quantitative technique comparatively to ELISA, its mainadvantage is the absence of interference of plasma lipids. FIG. 4Asymbols are as follows. N: umbilical cord serum of neonates (38 and 31weeks of gestation); A: plasma of an adult (20 years-old). FIG. 4Brefers to plasma from different times of human gestation (25 weeks, 36weeks and 38 weeks); nursing babies from different periods of lactation(2 and 8 months-old) and children of different ages (1, 5 and 10years-old).

ApoA-IV Levels in Umbilical Cord Serum of Human Neonates from Alcoholicand Control Mothers According to the Distribution's Body Weight.

TABLE 1 shows body weight, and levels of apoA-IV, triacylglycerides, andcholesterol in umbilical cord serum of neonates of 36 to 42 weeksgestation. ApoA-IV of these samples was quantified through ELISAsandwich (FIG. 3). To determine triacylglycerides and cholesterol inserum, the commercial kits were used (Medical Analysis Systems andBoehringer Mannheim GmbH respectively). Ethanol in utero significantlyreduced the apoA-IV in umbilical cord serum at birth, regardless of theneonate's body weight. This result could be related with morphologicaland functional disorders of the intestine found by the inventors in theexperimental foetus (from rat) affected by alcohol ingested by themother, probably linked to ethanol present in amniotic fluid andintestinal lumen of the foetus during gestation. Data give mean±SEM of nindividual. The Mann-Whitney U test was applied. Neonates C=neonates ofcontrol mothers. Neonates A=neonates of alcoholic mothers. NS=notsignificant.

FIG. 5 presents data for apoA-IV in serum (TABLE 1) according toneonates' weight distribution. Neonates from alcoholic mothers alwayshad less apoA-IV than control neonates (specially in neonates weighingbetween 2.5 and 3.5 Kg). Data give mean±SEM of n individual. Filledbars=neonates of control mothers. Open bars=neonates of alcoholicmothers. TABLE 1 Body weight, apoA-IV, triacylglycerides, andcholesterol in umbilical cord serum of neonates of 36 to 42 weeksgestation. Neonates C Neonates A n = 139 n = 78 p-Values Body weight (g)3140 ± 29  3220 ± 32  NS ApoA-IV (mg/100 ml) 1.66 ± 0.09 1.14 ± 0.11<0.001 triacylglycerides 40.43 ± 1.51  41.22 ± 2.17  NS (mg/100 ml)Cholesterol (mM) 1.75 ± 0.04 1.78 ± 0.05 NSWestern Blot Assay to Detect ApoA-IV in Umbilical Cord Serum of HumanNeonates from Alcoholic and Control Mothers.

FIG. 6 shows a Western blot of apoA-IV analysing serum of some neonatesbabies of control and alcoholic mothers with different lengths ofgestation. It is observed that the blot intensity in serum of offspringfrom alcoholic group is less detectable than in serum of controlneonates with the same length of gestation. These results complementdata of TABLE 1 and FIG. 5. Cord serum in neonates of 35, 36, 40 and 41weeks gestation. C=neonates from control mothers. A=neonates fromalcoholic mothers.

ApoA-IV Levels in Plasma of Coeliac Children

TABLE 2 shows the concentration in plasma of apoA-IV, triacylglyceridesand cholesterol in active coeliac children (with gluten intake) between1 and 6 years-old and controls of the same age. ApoA-IV of these sampleswere quantified through ELISA sandwich (FIG. 3). To determinetriacylglycerides and cholesterol in plasma, the commercial kits wereused (Medical Analysis Systems and Boehringer Mannheim GmbHrespectively). Triacylglyceride values were significantly higher incoeliac children than controls. Cholesterol values were significantlylower in coeliac children than controls. ApoA-IV values in coeliacchildren were below values for controls, although no significantdifferences were found between groups. Data given are mean±SEM of nindividual. The Mann-Whitney U test was applied. NS=not significant.TABLE 2 ApoA-IV, triacylglycerides and cholesterol in plasma of activecoeliac children (between 2 and 6 years old) and controls of the sameage. Coeliacs Controls n = 40 n = 173 p-Values ApoA-IV (mg/100 ml) 7.87± 0.80 9.58 ± 0.70 NS Triacylglycerides 89.53 ± 6.81  55.93 ± 2.54 <0.01 (mg/100 ml) Cholesterol (mM) 3.23 ± 0.11 3.97 ± 0.26 <0.01Western Blot Assay to Detect ApoA-IV in Plasma of Coeliac Children atDifferent Moments of their Disease

Western blot assay of plasma apoA-IV in coeliac children at differentmoments of their disease is presented in FIG. 7. In FIG. 7A plasmasamples of coeliac and control children and 80 ng of purified apoA-IVwas loaded. The coeliac child in active phases of their disease [withpositive serum markers (+): IgA-AGA and IgA-AEA antibodies] had lessintense apoA-IV blot than the same children nourished with a gluten-freediet (Ø), and significant lower than figures for controls of the sameage (FIG. 7B) (data as arbitrary units of six given individual). FIG. 7symbols are as follows. C=control children. Ce=coeliac children. [+ or++]=the coeliac children in active phases of their disease with positiveserum markers (IgA-AGA and IgA-AEA antibodies). [Ø]=the same childrennourished with a gluten-free diet (treated-coeliac). In FIG. 7Barbitrary units are give as the mean±SEM for n=6 individual (C and Ce).The Mann-Whitney U test was applied (p<0.01**, and p<0.1* aresignificant differences versus C).

The lower apoA-IV levels in plasma assayed by Western blot of coeliacchildren reveals a disorder in the metabolism of this protein, probablyrelated to the atrophy of the intestinal mucosa and malnutrition ofthese children. Coeliac children aged between 1 and 6 have a disturbancein lipid metabolism characterised by a drop in the plasma concentrationof cholesterol and apoA-IV and a rise in plasma triacylglycerides. Thesedisturbances may be directly related to intestinal malabsorption andmalnutrition associated with coeliac disease.

Western Blot Assay to Detect ApoA-IV in Plasma of Surgical PatientsParenterally Nourished

Western Blot assay of plasma apoA-IV in parenterally nourished patients(different sex and age) after abdominal surgery (colon resection) werepresent in FIG. 8. Plasma samples of every patient were loaded as inFIG. 8A. Days of study were: three days prior to surgery (overnightfasting); during surgery (between 24 and 48 hours minimum of fast); and2, 4 and 7 days after surgery (receiving parenteral nutrition support).FIG. 8B shows arbitrary units quantified as percentage in relation tothe blot of an internal control (plasma from a healthy youngadult—overnight fasted) loaded in the same gel. These results clearlyshowed significant, successive and profound changes in the blot ofplasma apoA-IV along days of individual submitted to parenteralnutrition. FIG. 8A symbols are as follows: −3=three days prior tosurgery; 0=during surgery; +2, +4, +7=2, 4 and 7 days after surgery. InFIG. 8B arbitrary units were quantified as percentage in relation to theblot of an internal control. Data give mean±SEM of n=21 individual. TheWilcoxon test was applied (p<0.001*** means significant differencesversus preoperative values of a given patient, −3).

Western Blot Assay to Detect ApoA-IV in Plasma of Surgical PatientsFasted and in Nourished in Successive Enterally Diet

Results in parenteral-enterally nourished patients after abdominalsurgery (gastric resection) correspond to data of FIG. 9. FIG. 9Apresents results of Western blot assay of plasma apoA-IV in successiveparenteral- and enterally nourished patient. The same criteria ofclinical selection was used that for patients of study of FIG. 8. Daysof study were: just before surgery (18 hours minimum of fast); justbefore the onset of oral nutrition (three days minimum of parenteralnutrition); and after four days receiving enteral nutrition (excludedfat diet). FIG. 9B shows arbitrary units quantified as percentage inrelation to the blot of an internal control (plasma from a healthy youngadult—overnight fasted) loaded in the same gel. These results clearlyshowed in this group of individual that there is an increase of the blotwhen diet change of the fast state to enteral nutrition in the samepatient. FIG. 9A symbols are as follows: 0=just before surgery; 0=justbefore the onset of oral nutrition and three-four days on parenterallynutrition (fast state); and 4=four days receiving only enteralnutrition. In FIG. 9B arbitrary units were quantified as percentage inrelation to the blot of an internal control. Data give mean±SEM of n=6individual.

1. A method of diagnosis of an intestine function disorder caused by anabnormal level of functionally effective absorptive intestinal mass in apatient, comprising submitting serum and/or plasma sample(s) from bloodof said patient to an immunoassay for assessing the amount of humanapolipoprotein apoA-IV, wherein said immunoassay comprises use ofspecific antibody anti-human apoA-IV obtained from a protein selectedfrom the group consisting of purified intestinal human apoA-IV, arecombinant human apoA-IV, and an active fragments of human apoA-IV. 2.The method according to claim 1, wherein the immunoassay comprises anELISA sandwich type immunoassay.
 3. The method according to claim 2,wherein the ELISA sandwich type immunoassay involves the pretreatment ofthe sample(s) with a clearing buffer containing pancreatic cholesterolesterase.
 4. The method according to claim 1, wherein the immunoassaycomprises a Western blot assay.
 5. The method according to claim 1,wherein the immunoassay comprises an immunodiffusion assay.
 6. Themethod according to any of the claims 1-5, wherein the intestinefunction disorder comprises one or more conditions selected from thegroup consisting of malabsorption, coeliac disease, villus atrophy,short bowel syndrome, chronic diarrhea and flatulence.
 7. The methodaccording to claims 1, wherein the intestine function disorder comprisesmalabsorption.
 8. The method according claim 1, wherein the intestinefunction disorder comprises coeliac disease.
 9. The method accordingclaim 1, wherein the intestine function disorder comprises villusatrophy.
 10. The method according to claim 1, wherein the intestinefunction disorder comprises short bowel syndrome.
 11. The methodaccording to claim 1, wherein the intestine function disorder compriseschronic diarrhea.
 12. The method according to claim 1, wherein theintestine function disorder comprises flatulence.
 13. An in vitro kitfor the diagnosis of an intestine function disorder caused by anabnormal level of functionally effective absorptive mass in a patient,comprising: (i) an specific antibody anti-human apoA-IV obtained from aprotein selected from the group consisting of purified intestinal humanapoA-IV, a recombinant human apoA-IV and active fragments of humanapoA-IV; and (ii) other materials and/or components for assessing theamount of human apolipoprotein apoA-IV by immunoassay.
 14. The kitaccording to claim 13, wherein the immunoassay is of the comprises anELISA sandwich type immunoassay.
 15. The kit according to claim 13,wherein the immunoassay comprises a Western blot assay.
 16. The kitaccording to claim 13, wherein the immunoassay comprises animmunodiffusion assay.
 17. The kit according to claim 13, wherein theintestine function disorder comprises one or more conditions selectedfrom the group consisting of malapsorption, celiac disease, villusatrophy, short bowel syndrome, chronic diarrhea and flatulence.